Transcription factors represent a group of molecules within the cell that function to connect extracellular signals to intracellular responses. Immediately after an environmental stimulus, these proteins which reside predominantly in the cytosol are translocated to the nucleus where they bind to the promoter regions of various genes and activate the transcription of the respective target gene. Certain transcription factors belong to a group of genes which are transcribed without any de novo protein synthesis allowing quick response times to external stumuli.
EGR-1 (also known as early growth response 1, Krox-24, sif/268, NGFI-A, cef5, TIS8 and GOS30) is the protein product of a gene in the group of immediate early genes which exhibit transcriptional induction within 30 minutes of mitogen stimulation. EGR-1 plays a major role in the regulation of signaling cascades within cells of the immune system (McMahon and Monroe, J. Leukoc. Biol., 1996, 60, 159-166) as well as specific cellular processes such as apoptosis (Liu et al., Cancer Gene Ther., 1998, 5, 3-28). Furthermore, it is currently believed that EGR-1 plays a role in regulating DNA and RNA synthesis by binding to the promoter of the inosine-5'-monophosphate dehydrogenase (IMPDH) type II gene. The enzyme encoded by this gene controls a critical step in the biosynthesis of guanine nucleotides (McMahon and Monroe, J. Leukoc. Biol., 1996, 60, 159-166).
As a transcriptional activator, EGR-1 binds a consensus sequence in the promoter of several genes including tumor necrosis factor (TNF), IL-2, ICAM-1, and CD44 in T- and B-lymphocytes. In addition, it also activates the genes for IGF-II, TGF-beta-1, PDGF alpha chain and thymidine kinase (McMahon and Monroe, J. Leukoc. Biol., 1996, 60, 159-166).
EGR-1 has been shown to regulate cellular growth and differentiation in a diverse set of cell types including macrophage, fibroblast, hepatocyte, neuronal, granulocyte, thymocyte, hematopoietic and T-cell lineages. Moreover, these effects have been shown to be very cell-type and tissue specific (Liu et al., Cancer Gene Ther., 1998, 5, 3-28).
Manifestations of altered EGR-1 gene regulation appear in both injury and disease states. EGR-1 upregulates cytokine expression involved in the inflammation process (McMahon and Monroe, J. Leukoc. Biol., 1996, 60, 159-166) and the level of EGR-1 protein itself goes up at the site of vascular occlusive lesions (Khachigian and Collins, Circ. Res., 1997, 81, 457-461).
To date, strategies aimed at inhibiting EGR-1 function have involved the use of antisense oligonucleotides and gene knockouts in mice.
Studies in which cells were treated with antisense oligonucleotides targeted to the 5' untranslated region of either mouse or human EGR-1, resulted in the inhibition of phorbol ester-induced differentiation of mouse HL-60 cells into macrophages (Nguyen et al., Cell, 1993, 72, 197-209). In addition, it has been demonstrated that EGR-1 protein levels are increased in the condition known as mesangioproliferative glomerulonephritis, and that antisense oligonucleotides to the translation initiation codon of rat EGR-1 inhibit the proliferation of rat glomerular mesengial cells (Rupprecht et al., Med Klin, 1997, 92, 68-73). Functional studies that used the same antisense oligonucleotide used by Nguyen et al, targeted to the 5' untranslated region of human EGR-1 to block EGR-1 protein expression in human melanoma cells confirmed that EGR-1 expression and function are essential for apoptosis induced by thapsigargin or ionizing radiation (Ahmed et al., J Biol Chem, 1996, 271, 29231-29237; Muthukkumar et al., Mol Cell Biol, 1995, 15, 6262-6272). Finally, Perez-Castillo et al. demonstrated that both lectin- and IL-2-induced proliferation of T-cells were blocked by treatment with antisense targeted to the start codon of rat EGR-1 (Perez-Castillo et al., J Biol Chem, 1993, 268, 19445-19450).
Gene knockout studies involving mice in which the EGR-1 gene was eliminated by homologous recombination showed no gross immunological defects in the animals. However, the reproductive capacity of female mice lacking the EGR-1 gene was compromised due to the lack of hormone production (Liu et al., Cancer Gene Ther., 1998, 5, 3-28).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of EGR-1. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting EGR-1 function.
Antisense oligonucleotides capable of inhibiting EGR-1 function can prove useful in a number of therapeutic and research applications and are thereby believed to provide a promising new pharmaceutical tool.